Spectrophotometers are used to measure the optical density of liquid samples placed in a cuvette, i.e., a liquid sample container having at least two parallel transparent walls. In a spectrophotometer measurement, a horizontal light beam from a light source passes through air and then into one of the parallel walls of the cuvette, then through the sample, then through the opposite parallel wall of the cuvette, and then through air where it is then detected by a light detector.
In contrast to horizontal light beam spectrophotometers, microplate readers are designed as vertical light beam photometers. In a microplate reader, a vertical beam of light is used to read the optical density of samples contained in the wells of multi-assay plates (MAPs) because the wells are arranged in rectangular arrays (e.g. 8.times.12). In such rectangular arrays, neighboring wells would be in the way of a horizontal light beam. In microplate readers, a vertical beam of light from a light source passes through air, but in contrast to a horizontal light beam, enters the sample directly at the air-sample interface. In a microplate reader, the light beam exits through the bottom of the multi-assay plate (MAP) and is then detected by a light detector.
When liquid samples are analyzed in a microplate reader, the light beam passes through the liquid sample at a meniscus formed at the interface between the liquid sample and the air above the liquid sample. Meniscus size and shape is determined by the physical surface properties of the MAP wells and the liquid sample contained within the well(s). Aqueous samples in MAP wells that have hydrophobic surfaces tend to form either a flat or a downwardly sloping (convex) meniscus. Aqueous samples in MAP wells that have hydrophilic surfaces, in contrast, tend to form upwardly sloping (concave) meniscus. The surface properties of the MAP wells have a direct effect on the meniscus size and shape and, ultimately, the optical pathlength through the liquid sample. Therefore, variability in pathlength through the sample is caused by any variability in the shape of the meniscus. In addition, the meniscus acts as a lens and refracts light depending on meniscus size and shape. Furthermore, refraction of light is dependent on wavelength of the light. Thus, it is not possible to completely correct for meniscus effect on the light transmission at a first wavelength by measuring the effect at a second wavelength.
Evaporation is another effect that causes several problems in photometric analysis. First, evaporation affects the concentration of the reactants as the volume of the liquid sample in the wells decreases. Second, due to the heat of evaporation, evaporation affects the steady-state temperature of the samples. Taking heat energy to change a liquid into a gas, evaporation will prevent the temperature of the sample from reaching the desired incubator temperature of the analysis chamber within the microplate reader. Third, different evaporation rates of a liquid sample within different wells of the MAP will cause the temperature of such liquid sample to vary from well-to-well. All of these three effects from evaporation result in inaccurate photometric analysis.
Evaporation is a particularly acute problem in the analysis of small volume samples (e.g. 200 .mu.l or less) in MAP wells, because of the large surface to volume ratio. Further, evaporation tends to be a serious problem for liquid samples having an appreciable vapor pressure at ambient temperature. Evaporation is further exacerbated at elevated temperatures which are sometimes needed in an analysis. As the temperature of the MAPs are raised, the rate of evaporation increases.
Attempts have been made to reduce the problem of evaporation. For example, evaporation can be reduced by saturating the air above the wells with the vapor of the volatile liquid, e.g. water. This is best achieved by placing a sealing cover over the MAP. Vapor, however will begin to condense on the MAP cover as the air space above the liquid becomes supersaturated at the temperature of the cover. Customarily, the liquid condenses as fine beads on the cover. The condensation scatters light and significantly affects the measurement of optical density with vertical beam photometers. The light scatter appears as an increase in optical density. Furthermore, the amount of condensation frequently is not identical from well to well, thereby causing variability and error in optical density measurements.
Thus, prior to the present invention, covers for MAPs have not eliminated the problems of evaporation. In addition, prior covers did not address the problems encountered by the meniscus effect. At most, these prior covers merely reduced the harmful effect due to evaporation, and provided a benefit of maintaining samples sterile (sterility is especially important in the analysis of samples comprised of mammalian cells in culture).
Currently, an antifogging agent (e.g. Molecular Devices Cat. No. R8005) coated onto the sample side of MAP covers has been used to minimize problems due to evaporation in MAPs. The antifogging agent is applied dropwise to the inner surface of a MAP cover and is spread with an applicator or sponge. A thin translucent irregular hydrophilic film results, which in turn minimizes light scatter caused by condensation. The application of an antifogging agent to a MAPs cover is an improvement over an untreated cover, however it has problems of its own. First, the antifogging film is translucent and scatters light rather than being completely transparent. Second, the translucent cover becomes increasingly translucent as the air above the wells becomes saturated with aqueous vapor.
Thus, prior to the present invention, existing covers for MAPs, even in combination with an antifogging agent, failed to eliminate the problems of evaporation and condensation in MAPs, and moreover, did not address, let alone eliminate, the problems due to the meniscus effect.
Another multi-assay plate cover in the arts is the Nunc Immunology TSP (Nunc No. 44597 available from Fisher Scientific (Pittsburgh, Pa.) as Cat. No. 12-555-143). This cover, made of polystyrene, has 96 hollow projections having an inside diameter of 2.41 millimeters (mm) at the proximal, top portion and are tapered to 2.00 mm internal diameter at the distal, bottom portion. The distal end of the projections are completely rounded, having a radius of curvature of approximately 1.0 mm. The outside diameter of the projections is 3.96 mm at the proximal, top portion and is tapered to 3.61 mm near the distal, bottom portion, and each projection extends about 10.5 mm into the corresponding wells of a 96-well multiassay plate. The cover is designed to be used with MAPs manufactured by NUNC having 96 cylindrical wells in 8.times.12 rectangular arrays with the central axes of the cylindrical wells spaced at 9.0 mm intervals and each well having an internal diameter of about 6.6 mm. Examples of such MAPs are NUNC Cat. Nos. 449824, 439454, 442404, 446612, and 430431.
Still another multi-assay plate cover in the art is the Falcon F.A.S.T. multi-assay plate cover, Cat. No. 3931, manufactured by Becton Dickenson & Co. (Oxnard, Calif.) and available from Fisher Scientific as Cat. No. 08-772-26. This MAP cover, made of polystyrene, has 96 solid projections about 1.26 cm long, having a diameter of about 1.6 mm at the proximal, top portion and have a polystyrene bead of about 3.8 mm at the distal, bottom portion. The F.A.S.T. cover is designed to be used with Falcon F.A.S.T. microplate MAPs having 96 cylindrical wells in 8.times.12 rectangular arrays (Falcon Cat. No 3933).
A major problem with such MAP covers is that they are not usable to read the optical density of samples contained at sample sites in MAPS. Specifically, the cross-sectional area of the light-transmitting portion of the projections of the Nunc TSP and Falcon F.A.S.T. covers are very narrow. These projections, therefore, are unable to transmit light to a substantial fraction of the cross-sectional area the wells of the 96-well MAPs. Additionally both the Nunc TSP and Falcon F.A.S.T. covers fit loosely on the MAPs with which they are compatible, allowing these covers to move with sliding motion at least 0.5 mm from side to side as the multi-assay plates, with covers, are placed into a microplate absorbance reader. Because of this sliding motion, the projections are able to move, at least partially, out of any light beam intended to pass down the long axis of the projections. Also, the narrowest internal diameter of the projections is insufficient to accommodate customary light beams wider than about 1.0 mm in diameter (allowing for .+-.0.5 mm of optical misalignment of the light beam with respect to the long axis of the projections). With such prior art covers, light passing down the long axis of the projections strikes the internal side edges of the projections thereby causing an error in the measurement of the relative amount of light transmitted through sample sites in the MAP. Also, the projections of such prior art covers are excessively long to be used with the MAPs with which they are compatible, such that the optical pathlength, through the samples in a MAP, would be less than 2.0 mm and in some cases less than 1.0 mm. Also, the distal bottom ends of the projections are extremely rough such that any light beam traveling through the projections would be scattered greatly so as to miss the photodetector placed below the MAP wells, thus causing error in determination of sample concentration.